The fields of life science research and pharmaceutical development are critically dependent upon highly selective and sensitive quantitative assays for a wide range of different biomolecules (such as proteins, antibodies, cytokines, receptors, enzymes, peptides, nucleic acids, hormones, and the like) in complex clinical or biological samples (such as blood, urine, tissue or cellular extracts, cell culture supernatants, bioprocess feedstreams, and the like). In typical samples (which may contain thousands of different molecular species) the analytes of interest may be present at extremely low concentrations (nanograms per milliliter or less), but the samples may be available only in very small quantities (microliters or less). The rapid growth in the field of biotechnology and the introduction of many potential new drug targets from genomic research have created an increasing demand for more rapid and efficient analytical methods, without any sacrifice in performance.
In order to simultaneously obtain high selectivity (the ability to measure one very specific molecule in a complex mixture) and high sensitivity (the ability to accurately quantify very small concentrations or amounts), a number of analytical methods have been developed which couple powerful molecular separations with extremely responsive detection methods.
One of the most widely used of these separation-based methods is the Enzyme-Linked Immuno-Sorbent Assay or ELISA. In ELISA, an antibody is immobilized on a solid phase support and exposed to a liquid sample, enabling any antigen (analytical target) to bind specifically to the antibody. Non-binding molecules in the sample are washed away. The solid phase with bound target can then be exposed to either antigen or a second antibody specific to the target that are labeled with a linked enzyme. After the non-binding labeled molecules are washed away, the solid phase is then exposed to enzyme substrate under controlled conditions so that the amount of colored or fluorescent enzyme product formed is proportional to the amount of label present, which can be used in turn to quantify the amount of target present in the original sample.
Currently in the fields of life science research and pharmaceutical development, ELISAs are done almost entirely using plastic (typically polystyrene) multi-well plates called microtiter plates or microplates. The wall of each well serves as both the solid phase for binding the antibody and antigen, as well as the container for the sample and reagents that are added. Liquid addition is done by pipetting, and washing is done by rapidly pipetting a wash solution in and out of the well. Readout of the enzyme product is done through the transparent plastic wells with an optical plate reader that measures either absorbance or fluorescence. This technique is quite simple, requires minimal specialized equipment and is very flexible in terms of the reagent systems and assay formats that can be used.
However, the microplate ELISA method suffers from a number of serious drawbacks. The most important is that the antibody is bound to the wall of the well, and thus the only way sample and reagent molecules can reach the surface to interact is by molecular diffusion. Diffusion is a relatively slow process over the potential path length of several millimeters found in a typical microplate well, and so after liquids are added for each step, the user must allow the plate to incubate for anywhere from 30 minutes to several hours to overnight to allow the binding reaction to approach equilibrium. This makes the total assay turnaround time quite long, typically on the order of 4 to 24 hours.
In addition, microplate ELISAs are subject to a high degree of variability, due to the critical techniques required. The pipetting must be done very accurately and consistently into each well, and timing between wells can be very important. Temperature variation between the inner and outer wells in a plate can lead to variability, as can jarring or vibration of the plates during incubation. Most operators are not as careful as required due to the tedium of the work, and assay coefficients of variation of 10 to 30% or more are not uncommon. Automation of microplate ELISAs using conventional liquid handling robotic equipment is possible, but is quite complex and often does not improve reproducibility. Users often find that such automated assays must be constantly monitored by a human operator to prevent problems.
A related set of highly selective separations are used in a micro-preparative mode to isolate the target from a complex sample in preparation for mass spectroscopy (MS), using either an ElectroSpray Interface (ESI) or Matrix Assisted Laser Desorption Interface (MALDI) to ionize the sample upon entry into the instrument. MS is unique in its ability to very rapidly provide comprehensive identity and structural information on analyte molecules with high sensitivity from very small volumes of sample. Because of the rich structural information MS gives about individual molecular species (especially proteins), complex samples must be fractionated or at least significantly simplified to enable a meaningful MS analysis to be performed. Purification methods are also needed when the target of interest is present in very small concentrations relative to other components in the sample, as is often the case in clinical or biological samples. Once the samples are separated into individual fractions or peaks, additional processing (such as concentration, desalting, enzymatic digestion and/or matrix addition) often must be performed to prepare the sample for analysis by the MS instrument.
In sample prep for MS, the target molecules are selectively bound to a surface by immobilized antibodies or other selective surface groups (such as ion exchange, reversed phase, hydrophobic interaction, affinity, and the like), and non-binding contaminants are washed away. Then the bound target is eluted (using for example salt, acid or organic solvent) for collection into a tube or on a surface spot for further analytical processing. It is also possible to immobilize an enzyme (such as a protease or glycosidase) to the packed bed to enable very rapid processing of the target molecule prior to further analysis. The amounts of target analyte required for MS are very similar to those required for detection using an ELISA.
Currently two separation methods are most often used as a front-end for MS and for two-dimensional gel electrophoresis and for gradient high performance liquid chromatography (HPLC). Both of these techniques are powerful and work reasonably well for comprehensively searching through all of the components in complex samples. However, these methods are not without problems. Two-dimensional gels, for example are labor-intensive, have many steps, and require many hours or even days to complete (compared to the analysis time of the MS, which is usually a matter of seconds). HPLC is sometimes not compatible with large proteins, and instrumentation systems with comparable throughput can be almost as expensive and complex as the MS itself. Sample carryover can also be an issue in high throughput applications.
Many different types of small-scale adsorption-based separation devices have been developed, and some are offered for use in MS sample preparation. Most have been adapted from devices designed for solid phase extraction (SPE) used in general analytical chemistry. One popular approach is the “spin column”, in which a small packed bed is suspended in a microcentrifuge tube, with samples and eluents driven through using a laboratory centrifuge. Some spin columns are also designed to be driven by a vacuum manifold. Spin columns are offered by a number of vendors in a range of common surface chemistries (reversed phase, ion exchange, metal chelate affinity). Although they are simple, spin columns suffer from the need to collect the final product in a test tube, then transfer it by pipette to the next step in the process or to the MS interface. These sample transfer steps can lead to significant losses, especially with dilute samples. Spin columns are poorly suited for automation. Also, most of the available spin columns are too large (typical bed volumes of 10 to 200 μL) for handling sample volumes in the low microliter range or below. It is also virtually impossible to control the flow rate through a spin column with any precision, which can reduce capture efficiency and reproducibility.
Perhaps the most popular approach to simplified sample preparation for MS is the use of modified pipette tips containing adsorbent materials. In the Millipore ZipTip product, a standard chromatographic adsorbent is embedded in a sponge-like polymer matrix in the end of the tip. The matrix enables flow by aspiration in a standard pipettor with little pressure drop. The company has also made this technology available in a 96-well plate format (ZipPlate) driven by a vacuum manifold, primarily for use in in-gel digestion and purification of 2D gel spots. Glygen has developed a tip with a flattened area at the end with the adsorbent particles embedded thermally on the inner surface, which can handle sample volumes as low as 1 to 10 μL. PhyNexus produces pipette tips containing affinity chromatography resins sandwiched between sealed-on screens in standard 200 and 1000 μL pipette tips. The tips produce final product in an elution volume of 10 to 15 μL. These pipette tip products are simple and convenient, but suffer from a number of drawbacks. If used with syringes or pipettors, it is very difficult to achieve sufficiently slow flow rates for complete binding, especially when affinity or antibody separations are used. As a result, multiple aspirate/dispense cycles are needed. This, in turn, leads to non-quantitative and/or non-reproducible capture of the bound target providing typical recoveries for proteins only in the 20 to 40% range. Like spin columns, pipette tips can only perform one separation step at a time, with some type of transfer operation required between steps, with likely concomitant sample loss. Flow through the pipette tip can only go in and out through the distal port, which limits the flexibility of operation.
A number of academic labs and companies have worked to integrate the separation and other processing steps or improve MS sensitivity through modifications to the MALDI plate itself. One example is the SELDI (Surface-Enhanced Laser Desorption Ionization) ProteinChip product from Ciphergen Biosystems. In this approach, various surface chemistries are incorporated into a spot on the plate to effect physical adsorption, ion exchange, or separations with affinity binding using antibodies or receptors. etc.). A small volume of sample is incubated on the spot, the non-binding materials washed off, and then matrix is added prior to analysis. The MALDI plate approaches are, of course, not amenable for use in electrospray MS. They are also limited to use with a single binding selectivity, so that other separation and preparation steps must be carried out elsewhere. The amount of sample that can be processed in this manner is also limited, so significant concentration is difficult to achieve.
A combined system approach has been developed by Intrinsic Bioprobes. The Mass Spectrometric ImmunoAssay (MSIA) technology developed by this company uses pipette tips incorporating a porous glass frit, onto which antibodies are immobilized. The bound antigens isolated from samples are eluted onto a MALDI plate for analysis. In other products, a pipette tip antibody-based separation device (using a porous glass monolith solid phase) is used in combination with enzymes (such as trypsin) immobilized on the MALDI plate. Gyros AB has developed a microfluidic system in the form of a compact disk (CD)-shaped device that incorporates several separation steps (including antibody affinity) driven by centrifugal force. The major application for this system are ELISA and sample preparation prior to MALDI MS. Bruker Daltonics has introduced the ClinProt system for purification prior to MALDI MS based upon robotic liquid handling and magnetic beads. Other integrated systems have some interesting advantages, but most of them require complex and expensive dedicated instrumentation for implementation.
Thus the field of biomolecule separation is one in which there is still room for improvement to overcome some of the limitations in prior art approaches and standard equipment. In particular, the use of the microtiter plate is less appropriate today given the sensitivity and speed desired by modern analytical biochemistry.